Amongst the compounds which are useful in the perfume and flavor industry, involving hydroperoxide cleavage possibly carried out via enzymatic reaction, the so called “green notes” include n-hexanal, hexan-1-ol, 2-(E)-hexen-1-al, 2-(E)-hexen-1-ol, 3-(Z)-hexen-1-ol (also known as pipol) and 3-(Z)-hexen-1-al, which are widely used in flavors, particularly fruit flavors, to impart a fresh green character. Furthermore, green notes are essential for fruit aroma and are used extensively in the aroma industry. The demand for natural green notes has grown to exceed their supply from traditional sources such as mint (Mentha arvensis) oil. This has motivated research efforts toward finding alternative natural ways of obtaining these materials.
The synthesis of green note compounds starts from free (polyunsaturated) fatty acids such as linoleic (9-(Z), 12-(Z)-octadecadienoic) and α-linolenic (9-(Z), 12-(Z), 15-(Z)-octadecatrienoic) acids. In nature, these acids are released from cell membranes by lipolytic enzymes after cell damage. Fatty acid 13-hydroperoxides are formed by the action of a specific 13-lipoxygenase (13-LOX) and are subsequently cleaved by a specific 13-hydroperoxide lyase (13-HPOL) into a C6 aldehyde and a C12 ω-oxoacid moiety. The aldehydes can subsequently undergo thermal isomerization and/or be reduced by dehydrogenase enzymes to produce other C6 products (i.e., green notes) such as alcohols (Hatanaka A. (1993) Phytochemistry 34: 1201-1218; Hatanaka A. et al. (1987) Chemistry and Physics of Lipids 44: 431-361).
Guava has been identified as an excellent source of freeze-stable 13-HPOL for use in this synthetic pathway. Guava 13-HPOL is currently used in an industrial process for the production of green notes (U.S. Pat. No. 5,464,761). In this process, a solution of the required 13-hydroperoxides is made from linoleic or linolenic acid (obtained from sunflower and linseed oils, respectively) using freshly prepared soybean flour as a source of 13-LOX. This solution is then mixed with a freshly prepared puree of whole guava (Psidium guajava) fruit as the source for 13-HPOL. The aldehyde products are then isolated by distillation. When the corresponding alcohols are required, fresh baker's yeast is added to the hydroperoxide solution before it is mixed with the guava puree. The yeast contains an active alcohol dehydrogenase enzyme that reduces the aldehydes to the corresponding alcohols as the aldehydes are formed by 13-HPOL.
There are a number of disadvantages to this industrial process. The principal disadvantage is the requirement of large quantities of fresh guava fruit. This means that the process has to be operated in a country where fresh guava fruit is cheaply and freely available. Even if such a site is found, availability is limited to the growing season of the fruit. Good quality guava fruit, for example, is only available for ten months of the year in Brazil.
A second disadvantage is that the desired enzyme activities are rather dilute in the sources employed. This means that relatively large amounts of soy flour (5%), guava puree (41%) and yeast (22%) have to be used in the process. The large volumes of these raw materials that are required for industrial production place physical constraints on the yields of green notes that can be achieved and raise the costs for the industrial process.
A third disadvantage is that it is a large-volume batch process, which, by its nature, does not make maximum use of the 13-HPOL enzyme's catalytic activity, is relatively labor-intensive and generates a large amount of residual organic material. The residual organic material must subsequently be transported to a compost farm or otherwise discarded.
To overcome some of the disadvantages of this industrial process, EP 1 080 205 discloses purified and recombinant guava 13-HPOL proteins, nucleic acids, expression systems, and methods of use thereof. However, upon using the recombinant guava 13-hydroperoxide lyase for producing C6-aldehydes, it turned out that the yield of products obtained with the recombinant guava 13-hydroperoxide lyase could still be optimized.
Thus, there is still a strong interest in providing 13-HPOL enzymes which allow the recombinant expression of the enzyme and which can be used to obtain a high yield of the desired product.
The above mentioned objects of the invention are solved by the modified 13-hydroperoxide lyase polypeptides according to claim 1. Preferred embodiments are represented by the subject matter of the sub-claims.